Sindbis virus (SB) is the prototype member of the alphavirus genus, a group which includes human pathogens such as eastern (EEE), western, and Venezuelan equine encephalitis viruses that are endemic in parts of the U.S.. The recent U.S. introduction of Aedes albopictus, a mosquito which includes humans among its preferred species for blood meals, and the isolation of EEE from this species in Florida, strongly suggest that future human epidemics of EEE and other aIphaviruses could be more frequent and significantly expanded. The long term goal of this research is to utilize a molecular genetic approach to understand the mechanisms by which specific attenuating mutations perturb viral replication in cells and alter pathogenesis in vivo. This approach will discern the underlying basis of wild-type virulence at the molecular level, will contribute to further understanding of alphavirus structure and biology, and will be directly applicable to the design of vaccines for the pathogenic alphaviruses. Previous experiments demonstrated that a conformational alteration of the SB glycoprotein spike occurred after interaction at the cell surface but prior to internalization of infecting virions. A glycoprotein mutation which strongly attenuated the virulence of SB also increased the efficiency with which the mutant accomplished the rearrangement. The 1st aim is to characterize this conformational change with respect to the identity and possible function of newly exposed domains on the altered structure. The 2nd aim is to examine the role of glycoprotein processing on virion structure and viability. Previous work established that SB virions containing the unprocessed precursor of the E2 glycoprotein (PE2) were not viable, that viable second-site mutants could be selected, and that these double mutants were uniformly attenuated in mice. Possible mechanisms underlying the loss of viability, resuscitation by second-site mutation, and attenuation will be explored. In the 3rd aim, 3-D image reconstruction with cryo-electron microscopy will be used to compare the native structure with those which contain PE2. Neutralization epitopes and epitopes exposed only on altered particles will be localized by imaging virion-Fab complexes. Aim 4 seeks to determine the in vivo pathogenesis effects related to the structure-function relationships delineated in aims 1-3 and to examine the influence on pathogenesis of mutations in the 5' untranslated region of the genome.